Completed on 18 Jan 2017 by John Hangasky. Sourced from http://biorxiv.org/content/early/2016/12/27/097022.
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These results do in fact indicate that LPMOs react with H2O2, but it is difficult to conclude at this point that H2O2 is the natural substrate for these enzymes. Are there estimates of the extracellular concentrations of H2O2? Although other oxidoreductases are secreted with LPMOs, would these enzymes be able to produce high enough concentrations of H2O2 that would effectively compete with O2?
Numerous enzymes utilize H2O2 and other reactive oxygen species as substrates, yet this would be the first example of an enzyme that generates a free hydroxyl radical as an intermediate. As hydroxyl radicals are extremely reactive, how does the observed activity account for the selective hydroxylation of only the C1 or C4 of the glycosidic linkage? The observation of four different protein residues being oxidized is indicative of the non-selective nature of hydroxyl radicals. Although the LPMO reaction with O2 is slower, it does not ultimately lead to enzyme inactivation or protein modification.
When AnGOX was used for in situ generation of H2O2, it appears the 10 minute and 30 minute time points for the first two AnGOX concentrations are similar, albeit slightly higher, to the control. These minor differences could be due to the O2 competition nature of the assay and glucose oxidase having a relatively low KM(O2) (~35 µM). Only after increased AnGOX concentrations and longer time points are taken, are the oxidized products significantly higher. At these time points, high concentrations of H2O2 are being produced.
John Hangasky and members of the Marletta Lab